Pulmonary arterial hypertension is a debilitating disease characterized by an increase in pulmonary vascular resistance leading to right ventricular failure and death. PAH with no apparent cause is termed primary pulmonary hypertension (“PPH”). Recently, various pathophysiological changes associated with this disorder, including vasoconstriction, vascular remodeling (i.e. proliferation of both media and intima of the pulmonary resistance vessels), and in situ thrombosis have been characterized (e.g., D'Alonzo, G. E. et al. 1991 Ann Intern Med 115:343-349; Palevsky, H. I. et al. 1989 Circulation 80:1207-1221; Rubin, L. J. 1997 N Engl J Med 336:111-117; Wagenvoort, C. A. & Wagenvoort, N. 1970 Circulation 42:1163-1184; Wood, P. 1958 Br Heart J 20:557-570). Impairment of vascular and endothelial homeostasis is evidenced from a reduced synthesis of prostacyclin (PGI2), increased thromboxane production, decreased formation of nitric oxide and increased synthesis of endothelin-1 (Giaid, A. & Saleh, D. 1995 N Engl J Med 333:214-221; Xue, C & Johns, R. A. 1995 N Engl J Med 333:1642-1644). The intracellular free calcium concentration of vascular smooth muscle cells of pulmonary arteries in PPH has been reported to be elevated.
The pathogenesis of pulmonary hypertension (“PH”) is a complex and multifactorial process. Pathologic changes of pulmonary arteries, which involve endothelial dysfunction, endothelial and smooth muscle cell proliferation, and increased vasoconstriction, decrease the lumen area of the pulmonary microvasculature, causing fixed elevation of pulmonary vascular resistance. Although these pathological features are common to all forms of human PH, the mechanisms responsible for this abnormal vascular proliferation are unknown. However, impairment of endothelial functions leading to an imbalance of vasodilator and vasoconstrictor influences is likely to play a central role in the initiation and progression of PH. Drugs that improve the endothelial function or restore the altered balance of endothelium-derived vasoactive mediators such as endothelin-1 receptor antagonists and prostacyclin analogues are used to treat this disease with moderate success. The NO/cGMP axis is also considered as a major target for the treatment of PH. Recently, the type 5 phosphodiesterase inhibitor sildenafil has been identified as a promising therapeutic agent for PH. The type 5 phosphodiesterase is the major cGMP-degrading phosphodiesterase in the pulmonary vasculature and is upregulated in PH. Sildenafil reduces right ventricular hypertrophy in chronic hypoxic mice, pulmonary arterial pressure in chronic hypoxic rats, and improves survival rate in rats with PH induced by monocrotaline injection. Short-term studies in patients with PH suggest that sildenafil is an effective pulmonary vasodilator.
On the other hand, recent pharmacological studies have suggested a role for the serine/threonine kinase Rho kinase in the development of PH. In vivo, intravenous or oral treatment with Rho kinase inhibitor (Y-27632 or fasudil) nearly normalizes the high pulmonary arterial pressure in chronically hypoxic rats, attenuates the development of chronic hypoxia-induced PH in mice, and reduces pulmonary arterial lesions in the model of monocrotaline-induced PH in rats (Abe K. et al. 2004 Circ. Res. 94:385-393; Fagan K. A. et al. 2004 Am. J. Physiol. Lung Cell. Mol. Physiol. 287:L656-L664; Nagaoka et al., 2004 Am. J. Physiol. Lung Cell Mol. Physiol. 287:L665-L672). In addition, inhaled Y-27632 or fasudil causes sustained and selective pulmonary vasodilation in monocrotaline-induced PH and in spontaneous PH in fawn-hooded rats, as well as in chronically hypoxic rats (Nagaoka et al. 2005 Am. J. Respir. Crit. Care Med. 171:494-499). Rho kinase is one of the main downstream effectors of the small G protein RhoA, which functions as a tightly regulated molecular switch that governs a wide range of cellular functions (Van Aelst & D'Souza-Schorey, 1997 Genes Dev. 11:2295-2322). In particular, Rho kinase phosphorylates the myosin phosphatase target subunit 1 (MYPT1) of smooth muscle myosin phosphatase at Thr-696, leading to the inhibition of its activity. This inhibition of smooth muscle myosin phosphatase activity is a primary mechanism of the Ca2+ sensitization of smooth muscle contraction. A large body of evidence has now been obtained regarding the important functions of RhoA in the vasculature, and RhoA has been shown to play a major role in the regulation of vascular cell processes such as actin cytoskeleton organization, contraction, gene expression, and differentiation. In vascular smooth muscle cells, RhoA has been shown to be regulated by the NO/cGMP pathways. RhoA is phosphorylated by cGMP-dependent protein kinase (PKG) (Sauzeau et al., 2000 J. Biol. Chem. 275:21722-21729). This phosphorylation prevents the translocation of active GTP-bound RhoA to the membrane, which is an obligatory step for the activation of its downstream effectors. Activation of the NO/cGMP pathway thus leads to the inhibition of RhoA-dependent functions, including actin cytoskeleton organization, Ca2+ sensitization of the contraction, and gene transcription (Sauzeau et al., 2000 J. Biol. Chem. 275:21722-21729; Gudi et al., 2002 J. Biol. Chem. 277:37382-37393). The beneficial effect of Rho kinase inhibitor on PH could be ascribed to multiple mechanisms, including its inhibitory effect on pulmonary vasoconstriction (Robertson et al., 2000 Br. J. Pharmacol. 131:5-9; Wang et al., 2001 Am. J. Respir. Cell Mol. Biol. 25:628-635; Nagaoka et al., 2004 Am. J. Physiol. Lung Cell Mol. Physiol. 287:L665-L672), the prevention of mechanical stress-induced expression of growth factors (Wilson et al., 1993 J. Cell Biol. 123:741-747), or the inhibition of Rho kinase-mediated mitogenic effects of serotonin (Liu et al., 2004 Circ. Res. 95: 579-586) and Rho kinase-mediated inhibition of nitric oxide synthase (Takemoto et al., 2002 Circulation 106: 57-62).
Current therapies for pulmonary hypertension are unsatisfactory. These typically involve calcium channel antagonists, prostacyclins, endothelin receptor antagonists and long-term anticoagulant therapy. However, each treatment has limitations and side effects.
Consequently there is a long felt need for a new and combined medicament for the treatment of PAH, preferably employing lower doses of the active agents, which exhibits fewer or no adverse effects (i.e., less toxicity) and a favorable profile in terms of effectiveness in patients in different stages of PAH.